PKC-activating compounds for the treatment of neurodegenerative diseases

ABSTRACT

The present invention relates to methods of activate an isoform of protein kinase C (PKC) for the treatment of neurological diseases including Alzheimer&#39;s disease and stroke using cyclopropanated or epoxidized derivatives of mono- and polyunsaturated fatty acids. The present invention also relates to methods of reducing neurodegeneration using cyclopropanated or epoxidized derivatives of mono- and polyunsaturated fatty acids.

This is a continuation of application Ser. No. 12/510,681, filed Jul.28, 2009 now U.S. Pat. No. 8,163,800, and claims the benefit of U.S.provisional patent application Ser. No. 61/084,172, filed on Jul. 28,2008, the disclosure of all of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods to activate anisoform of protein kinase C (PKC). The present invention also providesmethods for reducing neurodegeneration and for treatment of neurologicaldiseases including Alzheimer's disease and stroke.

BACKGROUND OF THE INVENTION

PKC Activators in Alzheimer's Disease, Stroke, and Depressive Disorders

Alzheimer's disease (AD) is a neurodegenerative disorder characterizedby the progressive decline of memory and cognitive functions. Dementiaassociated with AD is referred to as senile dementia of the Alzheimer'stype (SDAT) in usage with Alzheimer's disease. AD is characterizedclinically by progressive loss of memory, cognition, reasoning,judgment, and emotional stability that gradually leads to profoundmental deterioration and ultimately, death. Although there are manyhypotheses for the possible mechanisms of AD, one central theory is thatthe excessive formation and accumulation of toxic beta-amyloid (Aβ)peptides either directly or indirectly affects a variety of cellularevents and leads to neuronal damage and cell death. Selkoe, Neuron.1991; 6(4):487-98 1991; Selkoe, J Clin Invest. 2002; 110(10):1375-81.

AD is a progressive disorder with a mean duration of around 8-15 yearsbetween onset of clinical symptoms and death. AD is believed torepresent the seventh most common medical cause of death and affectsabout 5 million people in the United States. The prevalence is expectedto reach 7.7 million by 2030. About 1 in 8 people over the age of 65,13% of this population, have AD (Alzheimer's Association 2008Alzheimer's Disease Facts and Figures). AD currently affects about 15million people world-wide (including all races and ethnic groups) andowing to the relative increase of elderly people in the population itsprevalence is likely to increase over the next two to three decades. ADis at present incurable.

Protein kinase C (PKC) is one of the largest gene families of proteinkinase. Several PKC isozymes are expressed in the brain, including PKC,PKCβ1, PKCβII, PKCδ, PKCε, and PKCγ. PKC is primarily a cytosolicprotein, but with stimulation it translocates to the membrane. PKC hasbeen shown to be involved in numerous biochemical processes relevant toAlzheimer's disease. PKC activation also has a crucial role in learningand memory enhancement and PKC activators have been shown to increasememory and learning. Sun and Alkon, Eur J. Pharmacol. 2005; 512:43-51;Alkon et al., Proc Natl Acad Sci USA. 2005; 102:16432-16437. PKCactivation also has been shown to induce synaptogenesis in rathippocampus, suggesting the potential of PKC-mediated antiapoptosis andsynaptogenesis during conditions of neurodegeneration. Sun and Alkon,Proc Natl Acad Sci USA. 2008; 105(36): 13620-13625. Postischemic/hypoxictreatment with bryostatin-1, a PKC activator, effectively rescuedischemia-induced deficits in synaptogenesis, neurotrophic activity, andspatial learning and memory. Sun and Alkon, Proc Natl Acad Sci USA.2008. This effect is accompanied by increases in levels of synapticproteins spiniophilin and synaptophysin and structural changes insynaptic morphology. Hongpaisan and Alkon, Proc Natl Acad Sci USA. 2007;104:19571-19576. Bryostatin-induced synaptogenesis for long-termassociative memory is also regulated by PKC activation. Hongpaisan andAlkon, PNAS 2007. PKC also activates neurotrophin production.Neurotrophins, particularly brain-derived neurotrophic factor (BDNF) andnerve growth factor (NGF), are key growth factors that initiate repairand regrowth of damaged neurons and synapses. Activation of some PKCisoforms, particularly PKCε and PKCα, protect against neurologicalinjury, most likely by upregulating the production of neurotrophins.Weinreb et al., FASEB Journal. 2004; 18:1471-1473). PKC activators arealso reported to induce expression of tyrosine hydroxylase and induceneuronal survival and neurite outgrowth. Du and Iacovitti, J. Neurochem.1997; 68: 564-69; Hongpaisan and Alkon, PNAS 2007; Lallemend et al., J.Cell Sci. 2005; 118: 4511-25.

AD is also characterized by tau hyperphosphorylation. Tau is expressedmainly in the brain, where it regulates the orientation and stability ofmicrotubules in neurons, astrocytes and oligodendrocytes. In AD, normalsoluble tau is transformed into insoluble paired helical filaments. Thisis linked to the post-translational change in tau, primarily thehyperphosphorylation of tau by a number of protein kinases. Studies haveshown that synthetic Aβ promotes tau phosphorylation through activationof glycogen synthase kinase-3 GSK-3. Wang et al., Journal ofNeurochemistry. 2006; 98(4): 1167-1175. Activation of PKC has been shownto protects rat primary hippocampal neurons from Aβ-mediatedneurotoxicity, through inhibition of GSK-3β. Garrido et al., FASEB J.2002: 1982.

PKC also activates TNF-alpha converting enzyme (TACE, also known asADAM17), which is an enzyme that is involved in the proteolyticconversion of membrane-bound amyloid precursor protein (APP) to itsnon-pathogenic soluble form, known as soluble APP-alpha or sAPPα. Alkonet al., Trends in Pharmacological Sciences. 2007; 28(2): 51-60; Hurtadoet al., Neuropharmacology. 2001; 40(8): 1094-1102. These sAPPα-producingenzymes are referred to generically as alpha-secretases. Activation ofTACE by PKC also reduces cellular levels of pathogenic Aβ, which isproduced by cleavage of APP by the beta-secretase enzyme (BACE). This islikely due to the fact that the TACE cleavage site is within the Aβdomain of APP. Overexpression of PKCε has been shown to selectivelyincrease the activity of endothelin-converting enzyme (ECE), whichdegrades Aβ. Choi et al., Proc. Natl. Acad. Sci. USA. 2006; 103(21):8215-8220. In addition, sub-nanomolar concentrations of bryostatin and apotent synthetic analog (picolog), both PKC activators, were found tocause stimulation of non-amyloidogenic pathways by increasing TACE andthus lowering the amount of toxic Aβ produced. Khan et al., Proc. Natl.Acad. Sci. USA. 2009; 34(2):332-9.

Reduction of Aβ levels is a major therapeutic goal in Alzheimer'sdisease. It has been speculated that inhibition of Aβ formation by PKCactivators may be caused by competition of TACE and BACE for theircommon substrate, APP.

The strategy of PKC-mediated activation of α-secretases has theadvantage of three parallel beneficial consequences in AD: increasingproduction of sAPP-α and reducing Aβ, enhancing memory via PKC-mediatedphosphorylation of downstream substrates, and decreasing phosphorylationof tau through inhibition of GSK-3β.

AD patients already have reduced levels of PKCα/ε-mediatedphosphorylation of Erk1/2, a major downstream substrate of PKC. Khan andAlkon, Proc Natl Acad Sci USA. 2006; 103:13203-13207. In addition, Aβapplication to normal fibroblasts reduces PKC activity because Aβdirectly down-regulates PKC α/ε. PKC activators, especially thosespecific for PKC α/ε, would counteract the effect of Aβ and therebyreverse or prevent the Aβ-induced changes.

Stroke is a leading cause of disability and death in the United States,yet limited therapeutic options exist. Several PKC isoforms have beenshown to have a central role in mediating ischemic and reperfusiondamage following stroke. Studies with experimental stroke models, mousegenetics, and selective peptide inhibitors and activators havedemonstrated that PKCε is involved in induction of ischemic toleranceand prevents damage, while PKCδ and γ are implicated in injury.Takayoshi et al., Stroke. 2007; 38(2):375-380; and Bright et al.,Stroke. 2005; 36: 2781. One possible mechanisms for PKCε's protectiveischemic effect is that PKCε maintaining mitochondrial function via ERKactivity and by mediating adenosine-induced mitochondrial ATP-sensitivepotassium channels. Another potential mechanism is that PKCε elicits aneuroprotective effect via COX-2 induction. Kim et al., Neuroscience.2007; 145(3): 931-941. Prostaglandin E2 (PGE2), the product of COX-2activity, leads to neuroprotection in cerebral ischemia. As mentionedabove, postischemic/hypoxic treatment with bryostatin-1, a PKCactivator, effectively rescued ischemia-induced deficits insynaptogenesis, neurotrophic activity, and spatial learning and memory.Sun and Alkon, Proc Natl Acad Sci USA. 2008; 105(36): 13620-13625.

Circulating Aβ protein has been shown to be elevated in patients withacute ischemic stroke Circulating Aβ1-40 level was markedly elevated inischemic stroke patients, as compared to controls. Lee et al., Journalof Neural Transmission. 2005; 112(10): 1371-79. A strong positiveassociation between progressively accumulating vascular Aβ andaugmentations in arteriole and frontal cortex wall thickness AD patientsalso has been shown, suggesting that the continually progressingAβ-associated angiopathy, at the arteriolar level, harms the contractileapparatus and cerebral blood flow autoregulation, thereby making thedownstream capillaries vulnerable to damage. Stopa et al., Stroke. 2008;39:814.

In addition, some forms of stroke are caused by Aβ, such as thoseassociated with cerebral amyloid angiopathy, also known as congophilicamyloid angiopathy (CAA). This disorder is a form of angiopathy in whichthe same Aβ deposits as found in AD accumulate in the walls of theleptomeninges and superficial cerebral cortical blood vessels of thebrain. Amyloid deposition predisposes these blood vessel to failure,increasing the risk of a hemorrhagic stroke. CAA is also associated withtransient ischemic attacks, subarachnoid hemorrhage, Down syndrome, postirradiation necrosis, multiple sclerosis, leucoencephalopathy,spongiform encephalopathy, and dementia pugilistica.

Evidence suggests that PKCα and ε are the most important PKC isoforms ineliciting the aforementioned beneficial effects in AD, stroke, anddepressive disorders. Antisense inhibition of PKCα has been shown toblock secretion of sAPPα, while PKCε is the isozyme that mosteffectively suppresses Aβ production. Racci et al., Mol. Psychiatry.2003; 8:209-216; and Zhu et al., Biochem. Biophys. Res. Commun. 2001;285: 997-1006. Thus, isoform specific PKC activators are highlydesirable as potential anti-Alzheimer's drugs. Specific activators arepreferable to compounds such as bryostatin that show less specificitybecause non-specific activation of PKCδ or β could produce undesirableside effects.

Moreover, PKCε is also expressed at very low levels in all normaltissues except for brain. Mischak et al., J. Biol. Chem. 1993; 268:6090-6096; Van Kolen et al., J. Neurochem. 2008; 104:1-13. The highabundance of PKCe in presynaptic nerve fibers suggest a role in neuriteoutgrowth or neurotransmitter release. Shirai et al., FEBS J. 2008; 275:3988-3994). Therefore, effects of specific PKCε activators would belargely restricted to brain, and unlikely to produce unwanted peripheralside effects.

PUFAs as PKC Activators

Some PUFAs, such as arachidonic acid (see FIG. 1), have been known formany years to be natural activators of PKC. Docosahexaenoic acid (DHA)is also a known activator of PKC and has recently been shown to slow theaccumulation of Aβ and tau proteins associated with the brain-cloggingplaques and tangles implicated in AD. Sahlin et al., Eur J Neurosci.2007; 26(4):882-9.

Kanno et al. described effect of8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), anewly synthesized linoleic acid derivative with cyclopropane ringsinstead of cis-double bonds, on protein kinase C (PKC) activity. Journalof Lipid Research. 2007; 47: 1146-1156. DCP-LA activated PKCε, with agreater than 7-fold potency over other PKC isozymes. This indicates thatDCP-LA is highly specific for PKCε. This compound also facilitatedhippocampal synaptic transmission by enhancing activity of presynapticacetylcholine receptors on the glutamatergic terminals or neurons.However, DCP-LA requires relatively high concentrations to produce itsmaximal effect.

WO 2002/50113 to Nishizaki et al., discloses carboxylic acid compoundsand their corresponding salts having cyclopropane rings for LTP-likepotentiation of synaptic transmission or for use as acognition-enhancing drug or a drug to treat dementia. Their syntheticexamples disclose preparation of esters but their experimental resultsteach the use of free acids. The reason is that the carboxylic acidgroup of the fatty acid starting material would react with thediethylzinc used in the Simmons-Smith reaction. The methyl ester acts asa protecting group and may be cleaved off by hydrolysis or allowed toremain as needed.

The caveats with the prior art finding include the necessity ofadministering high concentrations of to achieve the foregoing effects,non-specific activation of PKC isoforms, or rapid metabolism andsequestration of unmodified PUFAs into fat tissues and other organswhere they are incorporated into triglycerides and chylomicrons. J.Pharmacobiodyn. 1988; 11(4):251-61. In addition use of unmodified PUFAswould have a myriad of adverse side effects. For example, arachidonicacid is a biochemical precursor to prostaglandins, thromboxanes, andleukotrienes, which have potent pro-inflammatory effects. This would beundesirable for treatment of Alzheimer's disease where the pathologylikely involves inflammation. Other essential fatty acids may alsopossess a multitude of other biological effects, including enhancementof nitric oxide signaling, anti-inflammatory effects, and inhibition ofHMG-CoA reductase, which would interfere with cholesterol biosynthesis.

Because of the limited existing options for treating both AD and stroke,new therapeutics that can selectively activate only the PKC isoformsthat elicit neuroprotection are needed.

PUFAs and MUFAs and Disease

A growing number of studies have suggested that omega-3 PUFAs can bebeneficial for other mood disturbance disorders such as clinicaldepression, bipolar disorder, personality disorders, schizophrenia, andattention deficit disorders. Ross et al., Lipids Health Dis. 2007; 18;6:21. There is an abundance of evidence linking omega-3 fatty acids,particularly docosahexaenoic and eicosapentaenoic acids, and a healthybalance of omega-3 to omega-6 fatty acids, to lowering the risk ofdepression. Logan et al., Lipids Health Dis. 2004; 3: 25. Levels ofomega-3 fatty acids were found to be measurably low and the ratio ofomega-6 to omega-3 fatty acids were particularly high in a clinicalstudy of patients hospitalized for depression. A recent studydemonstrated that there was a selective deficit in docosahexaenoic inthe orbitofrontal cortex of patients with major depressive disorder.McNamara et al. Biol Psychiatry. 2007; 62(1):17-24. Several studies havealso shown that subjects with bipolar disorder have lower levels omega-3fatty acids. In several recent studies, omega-3 fatty acids were shownto be more effective than placebo for depression in both adults andchildren with bipolar depression. Osher and Belmaker, CNS Neurosci Ther.2009; 15 (2):128-33; Turnbull et al., Arch Psychiatr Nurs. 2008;22(5):305-11.

Extensive research also indicates that omega-3 fatty acids reduceinflammation and help prevent risk factors associated with chronicdiseases such as heart disease, cancer, inflammatory bowel disease andrheumatoid arthritis. Calder et al., Biofactors. 2009; 35(3):266-72;Psota et al., Am J Cardiol. 2006; 98(4A):3i-18i; Wendel et al.,Anticancer Agents Med. Chem. 2009; 9(4):457-70.

Monounsaturated fatty acids also have been shown to be beneficial indisorders. There is good scientific support for MUFA diets as analternative to low-fat diets for medical nutrition therapy in Type 2diabetes. Ros, American Journal of Clinical Nutrition. 2003; 78(3):617S-625S. High-monounsaturated fatty acid diets lower both plasmacholesterol and triacylglycerol concentrations. Kris-Etherton et al., AmJ Clin Nutr. 1999 December; 70(6):1009-15.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structures of and of molecules contemplated for use according tothe present invention (BR-101 through BR-118).

FIG. 2 shows the results of an in vitro PKCε activation by BR-101(DCP-LA) and two less active derivatives, BR-102 and BR-103.

FIG. 3 shows activation of PKCε with various concentrations of BR-111(DHA-CP6 methyl ester); BR-114 (EPA-CP5 methyl ester); and BR-115(AA-CP4 methyl ester).

FIG. 4 shows activation of PKCε with various concentrations of othercyclopropanated and epoxidized fatty acid methyl esters: cyclopropanatedlinolenyl alcohol (BR-104); cyclopropanated linoleyl alcohol (BR-105);epoxystearic acid (BR-116); vernolic acid methyl ester (BR-117); andcyclopropanated vernolic acid methyl ester (BR-109).

FIG. 5 shows a time course of PKC activation by various concentrationsof bryostatin in H 19-7/IGF-IR rat hippocampal neurons.

FIG. 6 shows a time course of PKC activation in rat hippocampal primaryneurons by bryostatin and DCP-LA.

FIGS. 7 a and b depict decreased levels of intracellular (7 a) orsecreted (7 b) Aβ in neuro2a (N2A) cells exposed to PKC activatorsbryostatin, BR-101 (DCP-LA), or BR-111 (DHA-CP6).

FIG. 8 shows the effect of BR-111 (DHA-CP6) (0.1 to 10 μM) ondegradation of exogenously applied Aβ in SH-SY5Y neuroblastoma cells.

FIGS. 9 a-c depict effects of PKC activators PKC activators bryostatin,BR-101 (DCP-LA), and BR-111 (DHA-CP6) on TACE activity in N2aneuroblastoma cells transfected with human APPSwe/PS1D (9 a); theeffects of various concentrations of bryostatin on TACE activity in ratcortical primary neurons (9 b) and the effects of BR-111 (DHA-CP6) onTACE activity in rat cortical primary neurons (9 c).

FIG. 10 shows the activation of endothelin converting enzyme (ECE) byPKC activators bryostatin (0.27 nM), BR-101 (DCP-LA) (1 μM), BR-111(DHA-CP6) (1 μM), or ethanol in SH-SY5Y neuroblastoma cells.

FIGS. 11 a-b depict the effect of BR-101 (DCP-LA) and BR-111 (DHA-CP6)(1-100 μM) on cell survival and cell proliferation, respectively, ofSH-SY5Y neuroblastoma cells.

The present invention provides a method for activating PKCε usingcertain derivatives of polyunsaturated fatty acids (PUFA) ormonounsaturated fatty acids (MUFA). These compounds activate PKCε atnanomolar concentrations which makes them excellent candidates thr thetreatment of AD, stroke, and other neurological diseases in which PKCεis neuroprotective.

DEFINITIONS

A “fatty acid” is a carboxylic acid with an unbranched aliphatic chaincontaining from about 4 to 30 carbons; most long chain fatty acidscontain between 10 and 24 carbons. Fatty acids can be saturated orunsaturated. Saturated fatty acids do not contain any double bonds orother functional groups along the chain. Unsaturated fatty acids containone or more alkenyl functional groups, i.e., double bonds, along thechain. The term “polyunsaturated fatty acid” or “PUFA” means a fattyacid containing more than one double bond. There are three classes ofPUFAs, omega-3 PUFAs, omega-6 PUFAs, and omega-9 PUFAS. In omega-3PUFAs, the first double bond is found 3 carbons away from the lastcarbon in the chain (the omega carbon). In omega-6 PUFAs the firstdouble bond is found 6 carbons away from the chain and in omega-9 PUFAsthe first double bond is 9 carbons from the omega carbon.

PUFAs are also called “polyenoic fatty acids.” As used herein, the termPUFA includes both naturally-occurring and synthetic fatty acids. Amajor source for PUFAs is from marine fish and vegetable oils derivedfrom oil seed crops, although the PUFAs found in commercially developedplant oils are typically limited to linoleic acid and linolenic acid(18:3 delta 9,12,15).

A “cis-PUFA” is one in which the adjacent carbon atoms are on the sameside of the double bond.

The abbreviation X:Y indicates an acyl group containing X carbon atomsand Y double bonds. For example, linoleic acid would be abbreviated18:2.

A “methylene-interrupted polyene” refers to a PUFA having two or morecis double bonds separated from each other by a single methylene group.

A “non-methylene-interrupted polyene,” or “polymethylene-interruptedfatty acid,” refers to a PUFA having two or more cis double bondsseparated by more than one methylene group.

A “monounsaturated fatty acid” (MUFA) is a fatty acid that has a singledouble bond in the fatty acid chain and all the remaining carbon atomsin the chain are single-bonded. Exemplary MUFAs include oleic acid,myristoleic acid and palmitoleic acid.

A “cis-monounsaturated fatty acid” means that adjacent hydrogen atomsare on the same side of the double bond.

Conjugated fatty acids such as conjugated linoleic acid(9-cis,11-trans-octadecadienoic acid) possess a conjugated diene, thatis, two double bonds on adjacent carbons. Some evidence suggests thatconjugated linoleic acid has antitumor activity.

Exemplary PUFAs include lineoleic acid (9,12-octadecadienoic acid);linolenic acid (GLA; 6,9,12-octadecatrienoic acid); α-linolenic acid(9,12,15-octadecatrienoic acid); arachidonic acid(5,8,11,14-eicosatetraenoic acid); eicosapentanoic acid (EPA;5,8,11,14,17-eicosapentanoic acid); docosapentaenoic acid (DPA;7,10,13,16,19-docosapentaenoic acid); docosahexaenoic acid (DHA;4,7,10,13,16,19-docosahexanoic acid); and stearidonic acid(6,9,12,15-octadecatetraenoic acid).

As used herein, the term “cyclopropane” refers to a cycloalkane moleculewith the molecular formula C3H6, consisting of three carbon atoms linkedto each other to form a ring, with each carbon atom bearing two hydrogenatoms.

An “epoxide” refers to a cyclic ether with three ring atoms.

As used herein, a “PUFA derivative” refers to a PUFA, or alcohol orester thereof, in which at least one of the double bonds has beencyclopropanated or epoxidized.

As used herein, a “MUFA derivative” refers to a MUFA, or alcohol orester thereof, in which the double bond has been cyclopropanated orepoxidized.

“Selective activation” of PKCε means that the PUFA derivative compoundof the present invention activates PKCε to a greater detectable extentthan any other PKC isozyme. In specific embodiments, the PUFA derivativeactivates PKCε at least 1-fold, 2-fold or 5-fold over the other PKCisozymes as measured using e.g., the PKC activation assay describedherein. Upon activation, protein kinase C enzymes are translocated tothe plasma membrane by RACK proteins (membrane-bound receptor foractivated protein kinase C proteins). In general, upon activation,protein kinase C enzymes are translocated to the plasma membrane by RACKproteins. Other indicia of PKC activation include phosphorylation atspecific C-terminal serine/threonine residues byphosphatidylinositol-trisphosphate-dependent kinase (PDK1), with atleast two additional phosphorylations and/or autophosphorylations ofwell-conserved sequences in each enzyme of the PKC family. Activation ofPKC is described in Sun and Alkon, Recent Patents CNS Drug Discov. 2006;1(2):147-56.

“Neurodegeneration” refers to the progressive loss of structure orfunction of neurons, including death of neurons.

For purposes of the present invention, a “neurological disease” refersto any central nervous system (CNS) or peripheral nervous system (PNS)disease that is associated with the β-amyloidogenic processing of APP.This may result in neuronal or glial cell defects including but notlimited to neuronal loss, neuronal degeneration, neuronal demyelination,gliosis (i.e., astrogliosis), or neuronal or extraneuronal accumulationof aberrant proteins or toxins (e.g., Aβ).

One exemplary neurological disease is AD. Another exemplary neurologicaldisease is congophilic angiopathy (CAA), also referred to as cerebralamyloid angiopathy.

The term “Alzheimer's Disease” or “AD” refers to any condition where Aβdeposition will eventually in the cells of the central nervous system.In one, non-limiting embodiment, Aβ, particularly Aβ1-42, peptide isformed from the β-amyloidogenic metabolism of APP. AD may be heritablein a Familial manifestation, or may be sporadic. Herein, AD includesFamilial, Sporadic, as well as intermediates and subgroups thereof basedon phenotypic manifestations.

Another neurological disease is Down syndrome (DS). Subjects with DSinvariably develop (in their third or fourth decade) cerebral amyloid(Aβ) plaques and neurofibrillary tangles (NFTs), the characteristiclesions of AD. Recent studies have shown that the Aβ42 is the earliestform of this protein deposited in Down syndrome brains, and may be seenin subjects as young as 12 years of age, and that soluble Aβ can bedetected in the brains of DS subjects as early as 21 gestational weeksof age, well preceding the formation of Aβ plaques. Gyure et al.,Archives of Pathology and Laboratory Medicine. 2000; 125: 489-492.

As used herein, the term “subject” includes a mammal.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce untoward reactions when administered to a subject. Preferably,as used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of the federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. The term “pharmaceuticallyacceptable carrier” means a chemical composition with which the activeingredient may be combined and which, following the combination, can beused to administer the active ingredient to a subject and can refer to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered.

The terms “therapeutically effective dose” and “effective amount” referto an amount of a therapeutic agent that results in a measurabletherapeutic response. A therapeutic response may be any response that auser (e.g., a clinician) will recognize as an effective response to thetherapy, including improvement of symptoms and surrogate clinicalmarkers. Thus, a therapeutic response will generally be an ameliorationor inhibition of one or more symptoms of a disease or condition e.g.,AD. A measurable therapeutic response also includes a finding that asymptom or disease is prevented or has a delayed onset, or is otherwiseattenuated by the therapeutic agent.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

The present invention includes use of cyclopropanated and epoxidizedderivatives of PUFAs or MUFAs in which one, some, or all of the doublebonds are replaced by a cyclopropane group or an epoxide group. Theterminal function may be a free carboxylic acid, or a methyl ester,ethyl ester, or some other alkyl ester with an aliphatic or aromaticalcohol. This alcohol specifically may also include glycerol andderivatives thereof. Glycerol derivatives are biologically importantbecause the fatty acids are most frequently found conjugated to glycerolin the form of phosphatidylcholine, phosphatidylserine, or phosphatidicacids. For example, triacylglycerols are compounds in which the carboxylgroups of fatty acids are esterified to the hydroxyls of all threecarbons found in glycerol are referred to as triacylglycerols ortriglycerides.

The purpose of esterifying the carboxylic acid is to facilitatetransport across the blood-brain barrier by eliminating the negativecharge. The purpose of an alcohol group is also to facilitate transportacross the blood-brain barrier.

In one embodiment, the fatty acid which forms the basis for thecompounds used in the present invention is a polyunsaturated fatty acidhaving the following structure:CH₃(CH₂)₄(CH═CHCH₂)x(CH₂)yCOOHwherein X is between 2 and 6, and Y is between 2 and 6, and includemethylene- or polymethylene-interrupted polyenes. Exemplarypolyunsaturated fatty acids include linoleic acid, γ-linoleic,arachidonic acid, and adrenic acid having the following structures:Linoleic CH₃(CH₂)₄(CH═CHCH₂)₂(CH₂)₆COOHγ-Linolenic CH₃(CH₂)₄(CH═CHCH₂)₃(CH₂)₃COOHArachidonic CH₃(CH₂)₄(CH═CHCH₂)₄(CH₂)₂COOHAdrenic CH₃(CH₂)₄(CH═CHCH₂)₄(CH₂)₄COOHThese are omega-6 PUFAs.

In another embodiment, the fatty acid which forms the basis for thecompounds used in the present invention is a polyunsaturated fatty acidhaving the following structure:CH₃CH₂(CH═CHCH₂)x(CH₂)yCOOHwherein X is between 2 and 6, and Y is between 2 and 6 and includemethylene- or polymethylene-interrupted polyenes. Exemplarypolyunsaturated fatty acids include α-lineoleic acid, docosahexaenoicacid, eicosapentaenoic acid, eicosatetraenoic acid having the followingstructures:Alpha-Linolenic CH₃CH₂(CH═CHCH₂)₃(CH₂)₆COOHEicosatetraenoic CH₃CH₂(CH═CHCH₂)₄(CH₂)₅COOHEicosapentaenoic CH₃CH₂(CH═CHCH₂)₅(CH₂)₂COOHDocosahexaenoic CH₃CH₂(CH═CHCH₂)₆(CH₂)₂COOHThese are known as omega-3 PUFAs.

In a specific embodiment, the compound of the present invention is anester of a cis-PUFA, in which the hydroxyl group is replaced by analkoxy group, and in which at least one of the double bonds has beencyclopropanated. The starting material for this embodiment has thefollowing structures:CH₃(CH₂)₄(CH═CHCH₂)x(CH₂)yCOOR or CH₃CH₂(CH═CHCH₂)x(CH₂)yCOORwherein R is the alkyl group from an alcohol including monohydricalcohols and polyhydric alcohols including but not limited to methanol,ethanol, propanol, butanol, pentanol, glycerol, mannitol, and sorbitol.

In a further embodiment, the compound contains at least threecyclopropanated double bonds.

In another embodiment, the fatty acid which forms the basis for thecompounds used in the present invention is a monounsaturated fatty acidhaving the following structure:CH₃(CH₂)xCH═CH(CH₂)yCOOHwherein X and Y are odd numbers between 3 and 11.

Exemplary monounsaturated fatty acids that can be the basis for thecompounds used in the present invention include cis- andtrans-monounsaturated fatty acids such as oleic acid, elaidic acid,obtusilic acid, caproleic acid, lauroleic acid, linderic acid,myristoleic acid, palmitoleic acid, vaccenic acid, gadoleic acid, erucicacid, and petroselinic acid.

An ester according to the invention, means a monoester or a polyester.Esters of fatty acids include methyl, propyl, and butyl esters, and alsoesters resulting from more complex alcohols such as propylene glycol. Innon-limiting embodiments, R′ is straight or branched and includesmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tert-butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,and tetradecyl. An ester may also be formed from a fatty acid linked toa fatty alcohol in an ester linkage.

The ester can be a alcohol ester, including but not limited to analiphatic alcohol ester. In one embodiment, the alcohol ester is aglycerol ester. Glycerol esters of fatty acids include glycerol fattyacid ester, glycerol acetic acid fatty acid ester, glycerol lactic acidfatty acid ester, glycerol citric acid fatty acid ester, glycerolsuccinic acid fatty acid ester, glycerol diacetyl tartaric acid fattyacid ester, glycerol acetic acid ester, polyglycerol fatty acid ester,and polyglycerol condensed ricinoleic acid ester.

In another specific embodiment, the compound is an alcohol of a cis-PUFAin which at least one of the double bonds has been cyclopropanated. In afurther embodiment, the compound is an alcohol of a cis-PUPA whichcontains at least three cyclopropanated double bonds. These compoundsinclude but are not limited to linoleic alcohol dicyclopropane (BR-105),or linolenic alcohol tricyclopropane (BR-104). In this embodiment, R′can be a normal or branched chain alcohol or a phenolic alcohol.

In another embodiment, the compound of the present invention, thecompound is a cis-polyunsaturated fatty acid, or derivative thereof, inwhich at least one of the double bonds is replaced with an epoxyl group.In a further embodiment, the compound contains at least three epoxidizeddouble bonds.

In a specific embodiment, the compound is an epoxidized ester of acis-PUFA, including but not limited to a fatty alcohol ester. The esterscan be the same esters as described above for the cyclopropanated PUFAS.In a further embodiment the alcohol can be an aliphatic alcohol ester,such as glycerol.

In another specific embodiment, the compound is an epoxidizedcis-polyunsaturated fatty alcohol such as linoleic alcoholdicyclopropane or linolenic alcohol tricyclopropane. The alcohols can bethe same as described above for the cyclopropanated PUFAS.

In another embodiment, the compound includes cyclopropanated orepoxidized lipids derived from cis-monounsaturated fatty acids(cis-monoenoic acids), such as oleic acid, elaidic acid, elaidicalcohol, oleyl alcohol, and 1-monolinoleyl rac-glycerol. Exemplarycompounds include eliadic alcohol cyclopropane (BR-106), eliadic acidcyclopropane (BR-107), and oleyl alcohol cyclopropane (BR-108).

A further embodiment includes cyclopropanated lipids derived fromcis-monounsaturated fatty acids or unsaturated fatty acids, fatty acidesters, or fatty acid alcohols, containing one or more epoxide residues,such as vernolic acid methyl ester cyclopropane (e.g., BR-109).

In specific embodiments, the PUFAs which forms the basis of thecyclopropanated compounds used in the present invention include but arenot limited to arachidonic acid (AA), docosahexaenoic acid (DHA), andeicosapentaenoic acid (EPA). Exemplary compounds for use in the methodof the present invention include docahexaenonic acid methyl esterhexacyclopropane (BR-111); eicosapentaenoic acid methyl esterpentacyclopropane (BR-114); and arachidonic acid methyl estertetracyclopropane (BR-115).

In a further specific embodiment, the compound is a cyclopropanated PUFAderivative of docosahexaenoic acid having the following structure:

in which R is H or an alkyl group. In a specific embodiment, R is CH3(BR-111 or DHA-CB6 methyl ester ormethyl-3-(2-((2-((2-((2-((2-((2-ethylcyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)methyl)-cyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)propanoate.

In another specific embodiment, the PUFA derivative has the followingstructure:

This compound is BR-114 (EPA-CP5 or methyl4-(2((2((2-((2-ethylcyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)methyl)-cyclopropyl)butanoatemethyl ester).

In still another specific embodiment, the PUFA derivative has thefollowing structure:

This compound is BR-115 (AA-CP4 or methyl4-(2((2((2-((-pentylcyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)methyl)cyclopropyl)butanoatemethyl ester).

In yet another specific embodiment, the PUFA derivative has thefollowing structure:

in which R is H or an alkyl ester. In a specific embodiment, R is CII3.

Naturally cyclopropanated or epoxidized MUFAS or ester or alcoholderivatives thereof contemplated for use in the present inventioninclude malvenic acid, vernolic acid, and sterculic acid. An exemplarycompound is vernolic acid methyl ester (BR-117).

Methods of Synthesis

Fatty acids, and esters and alcohols thereof, can be obtained or madefrom purification from natural sources, e.g., fish oil, flaxseed oil,soybeans, rapeseed oil, or algae, or synthesized using a combination ofmicrobial enzymatic synthesis and chemical synthesis. As one example,fatty acid methyl esters can be produced by the transesterification oftriglycerides of refined/edible type oils using methanol and anhomogeneous alkaline catalyst.

Methods of cyclopropanation of double bonds in hydrocarbons are wellknown. As one example, the modified Simmons-Smith reaction is a standardmethod for converting double bonds to cyclopropanes. Tanaka andNishizaki, Bioorg. Med. Chem. Let. 2003; 13: 1037-1040; Kawabata andNishimura, J. Tetrahedron. 1967; 24: 53-58; and Denmark and Edwards, J.Org. Chem. 1991; 56: 6974. In this reaction, treatment of alkenes withmetal carbenoids, e.g., methylene iodide and diethylzinc, result incyclopropanation of the alkene. See also, Ito et al., Organic Syntheses.1988; 6:327. Cyclopropanation of methyl esters of was also effectedusing diazomethane in the presence of palladium (II) acetate ascatalyst. Gangadhar et al., Journal of the American Oil Chemists'Society. 1988; 65(4): 601-606.

Methods of epoxidation are also well known and typically involvereaction of fatty acids dioxiranes in organic solvents. Sonnet et al.,Journal of the American Oil Chemists' Society. 1995; 72(2):199-204. Asone example, epoxidation of PUFA double bonds can be achieved usingdimethyldioxirane (DMD) as the epoxidizing agent. Grabovskiy et al.,Helvetica Chimica Acta. 2006; 89(10): 2243-53.

Methods of Treatment

The present invention contemplates treatment of neurological diseasesassociated with pathogenic Aβ such as AD and stroke using the PUFAderivatives disclosed herein. The present invention also contemplatesprevention of neurological diseases associated with pathogenic Aβ usingthe PUFA derivatives disclosed herein. Without being limited to anyparticular mechanism, selective activation of PKCε may result inincreased activation of TACE, with a concomitant decrease in productionof Aβ. However, this appears to occur mainly in non-neuronal cells suchas fibroblasts. Activation of PKCε may also reduce thehyperphosphorylation of the pathogenic tau protein in AD. Activation ofPKCε may also induce synaptogenesis or prevent apoptosis in AD orfollowing stroke. Activation of PKCε may also protect rat neurons fromAβ-mediated neurotoxicity through inhibition of GSK-3β. PKC activatorsmay also counteract the effect of Aβ on the downregulation of PKC α/ε,and thereby reverse or prevent the Aβ-induced changes. Another possiblemechanism of action is the activation of Aβ-degrading enzymes such asendothelin-converting enzyme. The results of experiments presented inthe Examples suggest that this may be the mechanism of action.

Yet another mechanism may be by stimulation of PKC-coupled M1 and M3muscarinic receptors, which is reported to increase nonamyloidogenic APPprocessing by TACE. Rossner et al., Prog. Neurobiol. 1998; 56: 541-569.Muscarinic agonists rescue 3×-transgenic AD mice from cognitive deficitsand reduce Aβ and tau pathologies, in part by activating the TACE/ADAM17nonamyloidogenic pathway. Caccamo et al., Neuron. 2006; 49:671-682.Muscarinic receptor signaling is closely tied to PKC. Muscarinicreceptor mRNA is regulated by PKC and neuronal differentiation producedby M1 muscarinic receptor activation is mediated by PKC. Barnes et al.,Life Sci. 1997; 60:1015-1021; Vandemark et al., J. Pharmacal. Exp. Ther.2009; 329(2): 532-42.

Other disorders contemplated for treatment by the methods of the presentinvention include, mood disorders such as depressive disorders andbipolar disorder, schizophrenia, rheumatoid arthritis, cancer,cardiovascular disease, type 2 diabetes, and any other disorder in whichPUFAs or MUFAs have been shown to be beneficial, including but notlimited to those mention in the background.

Formulation and Administration

The PUFA derivatives may be produced in useful dosage units foradministration by any route that will permit them to cross theblood-brain barrier. It has been demonstrated PUFAs from plasma are ableto cross into the brain. Rapoport et al., J. Lipid Res. 2001. 42:678-685. Exemplary routes include oral, parenteral, transmucosal,intranasal, inhalation, or transdermal routes. Parenteral routes includeintravenous, intra-arteriolar, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, intrathecal, and intracranialadministration.

The compounds of the present invention can be formulated according toconventional methods. The PUFA derivative compounds can be provided to asubject in standard formulations, and may include any pharmaceuticallyacceptable additives, such as excipients, lubricants, diluents,flavorants, colorants, buffers, and disintegrants. Standard formulationsare well known in the art. See e.g., Remington's PharmaceuticalSciences, 20th edition, Mack Publishing Company, 2000.

In one embodiment, the compound is formulated in a solid oral dosageform. For oral administration, e.g., for PUFA, the pharmaceuticalcomposition may take the form of a tablet or capsule prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart. Liquid preparations for oral administration may take the form of,for example, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate.

As one example, the drug Omacor® contains concentrated combinations ofethyl esters of an omega-3 PUFAS. Each 1-g capsule contains at least 900mg of the ethyl esters of omega-3 fatty acids, primarily EPA (465 mg)and DHA (375 mg), according to the drug's label. Omacor® is administeredup to 4 times per day as 1-gram transparent soft gelatin capsules filledwith light-yellow oil. A similar composition can be used to administerthe PUFA compounds of the present invention, although the presentinvention contemplates use of a lower dose of the PUFA derivatives.Stable wax-ester formulations of PUFAs have also been described bytransesterification of stoichiometric amounts of ethyl esters enrichedwith n-3 PUFA and long-chain alcohols (18-22 carbon atoms) bytransesterification of stoichiometric amounts of ethyl esters enrichedwith n-3 PUFA and long-chain alcohols (18-22 carbon atoms). Goretta etal., Lebensmittel-Wissenschafi und-Technologie. 2002; 35(5): 458-65.

In another embodiment, the PUFA compound is formulated for parenteraladministration. The compound may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions, dispersions, or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents.

In a specific embodiment, the PUFA derivatives of the present inventionare administered with a hydrophobic carrier. Hydrophobic carriersinclude inclusion complexes, dispersions (such as micelles,microemulsions, and emulsions), and liposomes. Exemplary hydrophobiccarriers are inclusion complexes, micelles, and liposomes. Theseformulations are known in the art (Remington's: The Science and Practiceof Pharmacy 20th ed., ed. Gennaro, Lippincott: Philadelphia, Pa. 2003).The PUFA derivatives of the present invention may be incorporated intohydrophobic carriers, for example as at least 1, 5, 10, 20, 30, 40, 50,60, 70, 80, or 90% of the total carrier by weight. In addition, othercompounds may be included either in the hydrophobic carrier or thesolution, e.g., to stabilize the formulation.

In addition to the formulations described previously, the PUFAderivative may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

In another embodiment, the PUFA derivative can be delivered in avesicle, particularly a micelle, liposome or an artificial LDL particleas described in U.S. patent application Ser. No. 11/648,808 to Alkon etal.

The doses for administration may suitably be prepared so as to deliverfrom 1 mg to 10 g, preferably from 10 mg to 1 g and very preferably from250 mg to 500 mg of the compound per day. When prepared for topicaladministration or parenteral formulations they may be made in formulaecontaining from 0.01% to 60% by weight of the final formulation,preferably from 0.1% to 30% by weight, and very preferably from 1% to10% by weight. The optimal daily dose will be determined by methodsknown in the art and will be influenced by factors such as the age ofthe patient and other clinically relevant factors.

Combination Drug Therapy

The PUFA compound can be used to treat patients with AD or otherneurological disorders associated with Aβ in combination with otherdrugs that are also used to treat the disorder. Exemplary non-limitingpharmacological agents approved in the United States for the treatmentof AD include cholinesterase inhibitors such as Aricept® (donepezil),Exelon® (rivastigmine), Reminyl® (galantamine), and NMDA receptorantagonists such as Namenda® (memantine). Other potential therapeuticagents include protease inhibitors (see e.g., U.S. Pat. Nos. 5,863,902;5,872,101; inhibitors of Aβ production such as described in U.S. Pat.Nos. 7,011,901; 6,495,540; 6,610,734; 6,632,812; 6,713,476; and6,737,420; modulators of Aβ aggregation, described in 6,303,567;6,689,752; and inhibitors of BACE such as disclosed in U.S. Pat. Nos.6,982,264; 7,034,182; 7,030,239. Exemplary drugs used for the treatmentof stroke include aspirin, anti-platelet medications such as tissueplasminogen activator or other anticoagulants.

In a particular embodiment, the present invention contemplatescombination therapy with other PKC activators, including but not limitedto benzolactam macrocyclic lactones. Bryostatin-1 is a macrocycliclactone that has been shown to modulate PKC and result in an increase incleavage of APP by TACE into the non-amyloidogenic pathway. Bryostatinwas able to increase the duration of memory retention of the marine slugHermissenda crassicornis by over 500%, and was able to dramaticallyincrease the rate of learning in rats. See U.S. patent application Ser.No. 10/919,110; Kurzirian et al., Biological Bulletin. 2006; 210(3):201-14; Sun and Alkon, European Journal of Pharmacology. 2005; 512(1):43-51. Other non-limiting PKC activators are described in pending U.S.patent application Ser. No. 12/068,742 to Alkon et al.

Combinations with drugs that indirectly increase TACE, such as byinhibiting endogenous TACE inhibitors or by increasing endogenous TACEactivators. An alternative approach to activating PKC directly is toincrease the levels of the endogenous activator, diacylglycerol.Diacylglycerol kinase inhibitors such as6-(2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one(R59022) and[3-(2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quinazolinone(R59949) enhance the levels of the endogenous ligand diacylglycerol,thereby producing activation of PKC. Meinhardt et al. (2002) Anti-CancerDrugs 13: 725.

Still another embodiment is combination therapy with BACE inhibitors.BACE inhibitors are known and include CTS-21166, owned by CoMentis Inc.,which has shown positive results in a human clinical trial. Other BACEinhibitors are described in published International PCT applicationWO2007/019080 and in Baxter et al., Med. Chem. 2007; 50(18): 4261-4264.

Compounds used in combination therapy can be administered in the sameformulation as the PUFA compound of the present invention, wherecompatible, or can be administered in separate formulations.

Evaluation of Treatment

Evaluation of treatment with the PUPA derivatives of the presentinvention can be made by evaluation improvement in symptoms or clinicalsurrogate markers of the disease. For example, improvement in memory orcognitive skills in a treated AD subject may suggest that there is areduction of pathogenic Aβ accumulation. Examples of cognitivephenotypes include, but are not limited to, amnesia, aphasia, apraxiaand agnosia. Examples of psychiatric symptoms include, but are notlimited to, personality changes, depression, hallucinations anddelusions. As one non-limiting example, the Diagnostic and StatisticalManual of Mental disorders, 4th Edition (DSM-IV-TR) (published by theAmerican Psychiatric Association) contains criteria for dementia of theAlzheimer's type.

Phenotypic manifestations of AD may also be physical, such as by thedirect (imaging) or indirect (biochemical) detection of Aβ plaques. Invivo imaging of Aβ can be achieved using radioiodinated flavonederivatives as imaging agents (Ono et al., J Med. Chem. 2005;48(23):7253-60) and with amyloid binding dyes such as putrescineconjugated to a 40-residue radioiodinated A peptide (yielding 1251-PUT-A1-40), which was shown to cross the blood-brain barrier and bind to Aβplaques. Wengenack et al., Nature Biotechnology. 2000; 18(8): 868-72.Imaging of Aβ also was shown using stilbene [11C]SB-13 and thebenzothiazole [11C]6-OH-BTA-1 (also known as [11C]PIB). Verhoeff et al.,Am J Geriatr Psychiatry. 2004; 12:584-595.

Quantitation of Aβ (1-40) in the peripheral blood has been demonstratedusing high-performance liquid chromatography coupled with tandem massspectrometry in a linear ion trap. Du et al., J Biomol Tech. 2005;16(4):356-63. Detection of single Aβ protein aggregates in thecerebrospinal fluid of Alzheimer's patients by fluorescence correlationspectroscopy also has been described. Pitschke et al., Nature Medicine.1998; 4: 832-834. U.S. Pat. No. 5,593,846 describes a method fordetecting soluble Aβ. Indirect detection of Aβ peptide and receptor foradvanced glycation end products (RAGE) using antibodies also has beendescribed. Lastly, biochemical detection of increased BACE-1 activity incerebrospinal fluid using chromogenic substrates also has beenpostulated as diagnostic or prognostic indicator of AD. Verheijen etal., Clin Chem. 2006; 52:1168-1174.

Current measures for evaluation AD include observation of a clinicalcore of early, progressive and significant episodic memory loss plus oneor more abnormal biomarkers (biological indicators) characteristic ofAD, including atrophy (wasting) of the temporal lobe as shown on MRI;abnormal Aβ protein concentrations in the cerebrospinal fluid; aspecific pattern showing reduced glucose metabolism on PET scans of thebrain; and a genetic mutation associated with in the immediate family.

EXAMPLES Example 1 Synthesis of Fatty Acid Methyl Esters CyclopropanatedFatty Acid Methyl Esters

Synthesis of cyclopropanated fatty acids. Methyl esters ofpolyunsaturated fatty acids were cyclopropanated using the modifiedSimmons-Smith reaction using chloroiodomethane and diethylzinc (Tanakaet al., Bioorg. Med. Chem. Let. 2003; 13: 1037-40; Furukawa et al.,Tetrahedron. 1967; 53-58; Denmark et al., J. Org. Chem. 1991; 56:6974-81). All apparatus was baked at 60° C. for 1 hr and dried using aflame with dry nitrogen. A 100 ml 3-neck round bottom flask with astirring bar and a temperature probe was surrounded by an ice-dry icemixture and filled with 1.25 g (4.24 mmol) linoleic acid methyl ester ordocosahexaenoic acid methyl ester in 25 ml dichloromethane and bubbledwith N₂. A 1M solution of diethylzinc (51 ml, 54.94 mmol) in hexane wasadded anaerobically using a 24-inch-long 20-gauge needle and thesolution was cooled to −5° C. Diiodomethane (8.2 ml, 101.88 mmol) orchloroiodomethane (ClCH2I) was added dropwise, one drop per second, withconstant stirring. The rate of addition was decreased if necessary tomaintain the reaction mixture below 2° C. The reaction mixture becamecloudy during the reaction and an insoluble white zinc product wasliberated. The flask was sealed and the mixture was allowed to react for1 hr and then allowed to come to room temperature gradually over 2 hr.

To prevent the formation of an explosive residue in the hood,diethylzinc was not evaporated off. The mixture was slowly poured into100 ml of water under stirring to decompose any excess diethylzinc.Ethane was evolved. The mixture was centrifuged at 5000 rpm in glasscentrifuge tubes and the upper aqueous layer discarded. The whiteprecipitate was extracted with CH₂Cl₂ and combined with the organicphase. The organic phase was washed with water and centrifuged. Theproduct was analyzed by silica gel G TLC using hexane plus 1% ethylacetate and purified by chromatography on silica gel using increasingconcentrations of 1-10% ethyl acetate in n-hexane and evaporated undernitrogen, leaving the methyl ester as a colorless oil.

The Simmons-Smith reaction preserves the stereochemistry of the startingmaterials. Furukawa et al., Tetrahedron. 1967; 53-58. Docosahexaenoicacid methyl ester was converted into DHA-CP6 in 90-95% yield. Theproduct was a colorless oil with a single absorbance maximum at 202 nmin ethanol and no reaction with I₂. The IR spectrum showed cyclopropanering absorption at 3070 and 1450 cm⁻¹. Under the same conditions,eicosapentaenoic acid methyl ester was converted to EPA-CP5, andarachidonic acid methyl ester was converted to AA-CP4. Linoleic acidmethyl ester was converted to DCP-LA methyl ester which was identical toa known sample.

Hydrolysis of Methyl Ester.

The methyl ester (0.15 g) was dissolved in 1 ml 1N LiOH and 1 mldioxane. Dioxane and methanol were added until it became homogeneous andthe solution was stirred 60° overnight. The product was extracted inCH₂Cl₂ and centrifuged. The aqueous layer and white interface werere-extracted with water and washed until the white layer no longerformed. The product was evaporated under N₂ and purified bychromatography on silica gel. The product, a colorless oil, eluted in20% EtOAc in n-hexane. Its purity was checked by TLC in 10% EtOAc/hexaneand by C18 RP-HPLC using UV detection at 205 nm.

The epoxide groups can be introduced by conventional means, e.g., byoxidation of the appropriate alkene with m-chloroperbenzoic acid ort-butylhydroperoxide.

Other compounds synthesized include those depicted in FIG. 1 (BR-101through BR-118).

Example 2 Activation of Purified PKC Epsilon Using Docosahaexanoic Acid

Protein Kinase C Assay.

Recombinant PKC (1 ng of alpha or epsilon isoform) was mixed with theBR-101 (DCP-LA) in the presence of 10 micromolar histones, 5 mM CaCl₂,1.2 μg/μl phosphatidyl-L-serine, 0.18 μg/μl 1,2-dioctanoyl-sn-glycerol(DAG), 10 mM MgCl₂, 20 mM HEPES (pH 7.4), 0.8 mM EDTA, 4 mM EGTA, 4%glycerol, 8 μg/ml aprotinin, 8 μg/ml leupeptin, and 2 mM benzamidine.0.5 micro Ci [^(γ32)P]ATP was added. The incubation mixture wasincubated for 15 min at 37 degrees in a total volume of 10 microliters.The reaction was stopped by spotting the reaction mixtures on 1×2 cmstrips of cellulose phosphate paper (Whatman P81) and immediatelywashing twice for 1 hr in 0.5% H₃PO₄. The cellulose phosphate stripswere counted in a scintillation counter. In some experiments,phosphatidylserine, diacylglycerol, and/or calcium were removed.

DHA methyl ester was purchased from Cayman Chemical (Ann Arbor, Me.).PKC isozymes were from Calbiochem (San Diego, Calif.). Purified PKCε waspurchased from Calbiochem.

Results

PKC measurements using purified PKCε showed that, at the lowestconcentration tested (10 nM), compound BR-101 produced a 2.75-foldactivation of PKCε (FIG. 2). PKCε was not affected (data not shown).Compound BR-102 also selectively elicited activation of PKCε to about1.75 fold over unactivated PKCε. The effectiveness of these compounds inactivating PKCε at low concentrations suggests that they will be goodtherapeutic candidates.

Example 3 Activation of Purified or Cellular PKC Epsilon Using Other PKCActivators

Materials.

Culture media were obtained from K-D Medical (Columbia, Md.) orInvitrogen (Carlsbad, Calif.). Aβ1-42 was purchased from Anaspec (SanJose, Calif.). Polyunsaturated fatty acid methyl esters were obtainedfrom Cayman Chemicals, Ann Arbor, Mich. Other chemicals were obtainedfrom Sigma-Aldrich Chemical Co. (St. Louis, Mo.). PKC isozymes were fromCalbiochem (San Diego, Calif.). Purified PKCε was purchased fromCalbiochem.

Cell Culture.

Rat hippocampal H19-7/IGF-IR cells (ATCC, Manassas, Va.) were platedonto poly-L-lysine coated plates and grown at 35° C. in DMEM/10% FCS forseveral days until about 50% coverage was obtained. The cells were theninduced to differentiate into a neuronal phenotype by replacing themedium with 5 ml N₂ medium containing 10 ng/ml basic fibroblast growthfactor at 39° C. and grown in T-75 flasks at 37° C. Human SH-SY5Yneuroblastoma cells (ATCC) were cultured in 45% F12K/45% MEM/10% FCS.Mouse N2A neuroblastoma cells were cultured in DMEM/10% FCS withoutglutamine. Rat hippocampal neurons from 18-day-old embryonic

Sprague Dawley rat brains were plated on 12- or 96-well plates coatedwith poly-D-lysine (Sigma-Aldrich, St. Louis, Mo.) in B-27 neurobasalmedium containing 0.5 mM glutamine and 25 μM glutamate (Invitrogen,Carlsbad, Calif.) and cultured for three days in the medium withoutglutamate. The neuronal cells were grown under 5% CO₂ in an incubatormaintained at 37° C. for 14 days.

All experiments on cultured cells were carried out in triplicate unlessotherwise stated. All data points are displayed as mean±SE. BR-101(DCP-LA) was used as its free acid in all experiments, while BR-111(DHA-CP6), BR-114 (EPA-CP5), and BR-116 (AA-CP4) were used as theirmethyl esters.

Protein Kinase C Assay.

Rat hippocampal cells were cultured and scraped in 0.2 ml homogenizationbuffer (20 mM Tris-HCl, pH 7.4, 50 mM NaF, 1 μg/ml leupeptin, and 0.1 mMPMSF) and homogenized by sonication in a Marsonix micro-probe sonicator(5 sec, 10 W). To measure PKC, 10 μl of cell homogenate or purified PKCisozyme (purchased from Calbiochem) was incubated for 15 min at 37° C.in the presence of 10 μM histones, 4.89 mM CaCl₂, 1.2 μg/μlphosphatidyl-L-serine, 0.18 μg/μl 1,2-dioctanoyl-sn-glycerol, 10 mMMgCl₂, 20 mM HEPES (pII 7.4), 0.8 mM EDTA, 4 mM EGTA, 4% glycerol, 8μg/ml aprotinin, 8 μg/ml leupeptin, and 2 mM benzamidine. 0.5 μCi[^(γ-32)P]ATP was added and ³²P-phosphoprotein formation was measured byadsorption onto phosphocellulose as described previously. Nelson andAlkon, J. Neurochemistry. 1995; 65: 2350-57. For measurements ofactivation by BR-101 (DCP-LA) and similar compounds, PKC activity wasmeasured in the absence of diacylglycerol and phosphatidylserine, asdescribed by Kanno et al., and PKC δ, ε, η, and μ were measured in theabsence of added EGTA and CaCl₂, as described by Kanno et al., J. LipidRes. 2006; 47: 1146-50. Low concentrations of Ca²⁺ are used because highCa²⁺ interacts with the PKC phosphatidylserine binding site and preventsactivation. For measurements of bryostatin activation,1,2-diacylglycerol was omitted unless otherwise stated.

Results and Discussion

To determine their PKC isozyme specificity, the new compounds werepreincubated with purified PKC for five minutes and the PKC activity wasmeasured radiometrically. As shown for Example, 2, above, BR-101(DCP-LA) was an effective activator of PKCε at 10 μM but had relativelysmall effects on the other PKC isoforms (data not shown). At higherconcentrations BR-101 (DCP-LA) partially inhibited PKC (about 1-100 μM)and activated PKCγ (50-100 μM) (data not shown).

BR-111 (DHA-CP6), BR-114 (EPA-CP5), and BR-115 (AA-CP4), which arecyclopropanated derivatives of docosahexaenoic acid, eicosapentaenoicacid, and arachidonic acid, respectively, activated purified PKCε to asimilar extent (FIG. 3) The concentration needed to activate PKC wasapprox. 100 times lower than for BR-101 (DCP-LA), suggesting higheraffinity. Cyclopropanated linolenyl and linoleyl alcohols (BR-104 andBR-105), epoxystearic acid (BR-116), and vernolic acid methyl ester(BR-117) had little or no effect on PKC (FIG. 4). Cyclopropanatedvernolic acid methyl ester (BR-109) inhibited PKCε at concentrationsabove 1 μM (FIG. 4).

PKC activators that bind to the diacylglycerol binding site, includingbryostatin, gnidimacrin, and phorbol esters, produce a transientactivation of PKC activity, followed by a prolonged downregulation.Nelson et al., Trends in Biochem. Sci. 2009; 34: 136-45. This wasconfirmed in cultured rat hippocampal cells. Incubation of ratH19-7/IGF-IR cells with (0.04 nM and 0.2 nM) bryostatin produced a2-fold activation that lasted 30 min, followed by a 20% downregulationthat returned to baseline by 24 h (data not shown). In contrast, PKCexposed to DCP-LA remained elevated for at least four hours (FIG. 5).This sustained activation was only observed in primary neurons.

Even though bryostatin has a higher affinity for PKC than phorbol12-myristate 13-acetate (PMA)(EC50=1.35 nM vs. 10 nM), bryostatin wasmuch less effective than PMA at downregulating PKC. PKC activity isstrongly downregulated by phorbol ester at 8 h, while PKC inbryostatin-treated cells is at or near the baseline (data not shown).This difference may explain the increases in Aβ produced by PdBureported by da Cruz e Silva et al. J. Neurochem. 2009:108:319-30. Theseinvestigators applied 1 μM PdBu to cultured COS cells for 8 h andobserved an increase in Aβ. This increase was attributed todownregulation of PKC by the phorbol ester, which is consistent withthese results. Downregulation could not be measured for DCP-LA andrelated compounds.

Example 4 Effects of PKC Activators on Aβ Production and Degradation

Cell Culture.

Cell culture was performed as described above for Example 3.

Aβ Measurement and Cell Viability Assay.

Aβ was measured using an Aβ1-42 human fluorimetric ELISA kit(Invitrogen) according to the manufacturer's instructions. Results weremeasured in a Biotek Synergy HT microplate reader. AlamarBlue andCyQuant NF (Invitrogen) according to the manufacturer's instructions.

Results and Discussion

To measure the effects of PKCα activation on Aβ production, we usedmouse neuro2a (N2a) neuroblastoma cells transfected with humanAPPSwe/PSID, which produce large quantities of Aβ. Petanceska et al., J.Neurochem. 1996; 74: 1878-84. Incubation of these cells for 24 h withvarious concentrations of PKC activators. bryostatin, BR-101 (DCP-LA)and BR-111 (DHA-CP6) markedly reduced the levels of both intracellular(FIG. 7 a) and secreted (FIG. 7 b) Aβ. With bryostatin, which activatesPKC by binding to the diacylglycerol-binding site, the inhibition wasbiphasic, with concentrations of 20 nM or higher producing no neteffect. This may be explained by the ability of this class of PKCactivators to downregulate PKC when used at high concentrations. Incontrast, BR-101 (DCP-LA) and BR-111 (DHA-CP6), which bind to PKC'sphosphatidylserine site, showed monotonically increasing inhibition atconcentrations up to 10 to 100 μM with no evidence of downregulation athigher concentrations.

To determine whether the reduced levels of Aβ caused by PKC activatorswere due to inhibition of Aβ synthesis or activation of Aβ degradation,we applied BR-111 (DHA-CP6) (0.01 to 10 μM) and low concentrations (100nM) of exogenous monomeric Aβ-42 to cultured SH-SY5Y cells. Thisconcentration of Aβ is too low to produce measurable toxicity or celldeath. Since SH-SY5Y cells produce only trace amounts of Aβ, thisexperiment was an effective test of the ability of PKC activators toenhance Aβ degradation. By 24 h, most of the Aβ had been taken up by thecells and the concentration of Aβ in the culture medium wasundetectable. Addition of 0.01 to 10 μM DHA-CP6 to the cells reduced thecellular levels of Aβ by 45-63%, indicating that the PKCε activatorincreased the rate of degradation of exogenous Aβ (FIG. 8).

DHA-CP6, bryostatin, and DCP-LA had no effect on cell survival or onproliferation as measured by alamar Blue and CyQuant staining (FIGS. 11a and b), indicating that the reduction in Aβ production did not resultfrom cell proliferation or a change in cell survival.

Example 5 Effects of PKC Activators on TACE Activity

TACE Assay.

TACE was measured by incubating 5 μl cell homogenate, 3 buffer (50 mMTris-HCl 7.4 plus 25 mM NaCl plus 4% glycerol), and 1 μl of 100 μM TACEsubstrate IV (Aβz-LAQAVRSSSR-DPa) (Calbiochem) for 20 min at 37° in1.5-ml polypropylene centrifuge tubes (Jin et al., Anal. Biochem. 2002;302: 269-75). The reaction was stopped by cooling to 4° C. The sampleswere diluted to 1 ml and the fluorescence was rapidly measured (ex=320nm, em=420 nm) in a Spex Fluorolog 2 spectrofluorometer.

Results and Discussion

Previous researchers reported that PKC activators such as phorbol12-myristate 13-acetate produce large increases in TACE activity whichcorrelated with increased sAPPα and decreased Aβ, suggesting that TACEand BACE1 compete for availability of APP substrate, and that PKCactivators shift the competition in favor of TACE. Buxbaum et al., J.Biol. Chem. 1998; 273: 27765-67; Etcheberrigaray et al., Proc. Natl.Acad. Sci. USA. 2006: 103:8215-20. However, many of these earlierstudies were carried out in fibroblasts and other non-neuronal celltypes, which appear to respond differently to PKC activators thanneurons. For example, Etcheberrigaray et al. found that activation ofPKC in human fibroblasts by 10 pM to 100 pM bryostatin increased theinitial rate of α-secretase activity by 16-fold and 132-fold,respectively (Etcheberrigaray et al., Proc. Natl. Acad. Sci. USA. 2006).However, in human SH-SY5Y neuroblastoma cells, N2a mouse neuroblastomacells (FIG. 9 a), and primary neurons from rat hippocampus (FIGS. 9 b,c), PKC activators bryostatin, BR-101 (DCP-LA) and/or BR-111 (DHA-CP6)only produced small increases in TACE activity. This suggests that anyreduction of Aβ levels in neurons by PKC activators must be caused bysome other mechanism besides activation of TACE.

Example 6 Effects of PKC Activators on Endothelin-Converting EnzymeActivity

ECE Assay.

SH-S757 neuroblastoma cells were incubated with bryostatin (0.27 nM),BR-101 (DCP-LA) (1 μM), and BR-111 (DHA-CP6) (1 μM).Endothelin-converting enzyme (ECE) was measured fluorimetrically usingthe method of Johnson and Ahn, Anal. Biochem. 2000; 286: 112-118. Asample of cell homogenate (20 μl) was incubated in 50 mM MES-KOH, pH6.0, 0.01% C12E10 (polyoxyethylene-10-lauryl ether), and 15 μM McaBK2(7-Methoxycoumarin-4-acetyl [Ala7-(2,4-Dinitrophenyl)Lys9]-bradykinintrifluoroacetate salt) (Sigma-Aldrich). After 60 min at 37° C., thereaction was quenched by adding trifluoroacetic acid to 0.5%. The samplewas diluted to 1.4 ml with water and the fluorescence was measured atex=334 nm, em=398 nm.

Results and Discussion

Aβ can be degraded in vivo by a number of enzymes, including insulindegrading enzyme (insulysin), neprilysin, and ECE. Because PKCoverexpression has been reported to activate ECE (Choi et al., Proc.Natl. Acad. Sci. USA. 2006; 103: 8215-20), we examined the effect of PKCactivators on ECE. Bryostatin, BR-101 (DCP-LA), and BR-111 (DHA-CP6) allproduced a sustained increase in ECE activity (FIG. 10). Since ECE doesnot possess a diacylglycerol-binding C1 domain, this suggests that theactivation by bryostatin was not due to direct activation of ECE, butmust have resulted from phosphorylation of ECE or some ECE-activatingintermediate by PKC. This result also suggests that indirect activationECE by PKC activators could be a useful means of reducing the levels ofAβ in patients.

An advantage of compounds such as the PUPA derivatives of the presentinvention which specifically activate PKCε is that they produce lessdown-regulation than phorbol esters and similar 1,2-diacylglycerol (DAG)analogues. The biphasic response of PKC to DAG-based activators meansthat a PKC activator may reduce Aβ levels at one time point and increasethem at another. da Cruz e Silva et al., J Neurochem. 2009; 108:319-330. Careful dosing and monitoring of patients would be required toavoid effects opposite to those that are intended. Because of therelative inability of this new class of PKC activators to downregulatePKC, this problem can be avoided.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

What is claimed:
 1. A method of treating at least one neurodegenerativedisease and/or mood disorder comprising administering to a subject inneed thereof an effective amount of at least one compound chosen from acis-polyunsaturated fatty acid ester in which at least one of the doublebonds is replaced by a cyclopropyl group, wherein the at least oneneurodegenerative disease and/or mood disorder is chosen fromAlzheimer's disease, stroke, and depression.
 2. The method of claim 1,wherein the neurodegenerative disease is Alzheimer's disease.
 3. Themethod of claim 1, wherein the mood disorder is depression.